Potential Anti-Allergy and Immunomodulatory Properties of Lactococcus lactis LB 1022 Observed In Vitro and in an Atopic Dermatitis Mouse Model

Lactococcus lactis is a lactic acid bacterium and used in the dairy food industry. The ameliorating effects of Lactobacillus species on atopic dermatitis (AD) have been extensively studied, but the specific effect of L. lactis strains has not yet been investigated. In this study, the efficacy of L. lactis LB 1022, isolated from natural cheese, was evaluated using RAW 264.7, HMC-1 and HaCaT cell lines and an ovalbumin-sensitized AD mouse model. L. lactis LB 1022 exhibited nitric oxide suppression and anti-allergy and anti-inflammatory activity in vitro. Oral administration of L. lactis LB 1022 to AD mice significantly reduced the levels of IgE, mast cells, and eosinophils, and a range of T cell-mediated T helper Th1, Th2, and Th17-type cytokines under interleukin (IL)-10, transforming growth factor-β (TGF-β), thymus and activation-regulated chemokine (TARC), and thymic stromal lymphopoietin (TSLP). In addition, L. lactis LB 1022 treatment increased the concentration of short-chain fatty acids. Overall, L. lactis LB 1022 significantly modulated AD-like symptoms by altering metabolites and the immune response, illustrating its potential as candidate for use in functional food supplements to alleviate AD.


Measurement of β-Hexosaminidase and Histamine Release Assays
Inhibitory effects on release and activity from HMC-1 cells were performed as previously described [15]. Briefly, to the β-hexosaminidase inhibition assays, HMC-1 cells were seeded at 2 × 10 6 cells/well and added Tyrode's buffer (0.4 mM NaH 2 PO 4 , 1.8 mM CaCl 2 , 2.7 mM KCl, 5.6 mM glucose, 11.9 mM NaHCO 3 , and 137 mM NaCl; pH 7.2) and L. lactis LB 1022 (10 8 CFU/ml) for 30 min. Samples of 20 μl were then incubated at 37°C for 1 h with 30 μl of 1 mM p-nitrophenyl-N-acetyl-β-D-glucosaminide suspended in a 0.1 M citrate buffer. Finally, the samples were terminated to stop the reaction. The absorbance was measured at OD 405 using an Infinite M200 microplate reader (Nano-quant). Histamine assay was conducted using a histamine ELISA kit (Cayman Chemical Company, USA). The absorbance was measured at OD 405 with an Infinite M200 microplate reader (Nano-quant) and compared with a standard curve to identify the histamine levels.

Animal Studies
Female BALB/c mice (5 weeks old) were obtained from Central Laboratory Animal in Korea (Korea). The animals were maintained in hard-top cages (five mice per cage) under a 12-h light/dark cycle at a temperature of 22 ± 2°C and humidity of 50 ± 10%. The experiments were approved by the Animal Care Committee of Chung-Ang University (IACUC no. 2018-00022). The mice were distributed into three groups (n = 5 / group) as follows: control, OVA (Sigma-Aldrich; the AD-induced group), and L. lactis LB 1022 (AD + treatment with L. lactis LB 1022). To sensitive an AD immunological response and skin lesions, once a week, the hair on the dorsal skin of the mice was shaved. Six-week-old BALB/c mice were sensitized intraperitoneally with OVA (50 μg/ml) and alum (Sigma-Aldrich) at 1, 3, 5, and 7 weeks. The LB 1022 group received L. lactis LB 1022 (1 × 10 8 CFU) orally using a gavage for 8 weeks.

Dermatitis Scoring
The dermatitis score of AD on the dermal was scored twice a week as the sum of individual erythema, dryness, edema, and erosion/excoriation scores (0, none; 1, mild; 2, moderate; and 3, severe).

Real-Time Quantitative PCR Analysis
Total RNA was extracted from skin material by a RNeasy Mini Kit (Qiagen, Germany) and the cDNA was synthesized using a PrimeScript 1st strand cDNA Synthesis Kit (Takara, Japan) following the manufacturer's instructions and quantitatively analyzed with SYBR Green (Qiagen) using a QuantStudio 3 system. The 36B4 housekeeping gene was used as control. The primers used were presented in Table 1.

Histological Analysis
The dorsal skin tissue was collected and fixed with 10% formaldehyde solution and embedded in paraffin. Paraffin blocks were cut to a thickness of 4-5 μm and stained with hematoxylin and eosin solution (H&E; Sigma-Aldrich) for inflammation and Toluidine Blue solution (Sigma-Aldrich) for mast cells under a DM 4000B microscope (Leica Microsystem, Wetzlar).

Short-Chain Fatty Acid Analysis
To determine whether the SCFA levels are different between control, LB 1022, and OVA, stool samples were collected from each mouse at 8, 12, and 16 weeks and stored at −80°C. The samples were reconstituted in deionized water and passed through a membrane filter using Sep-Pak Vac cartridges (WAT054955; Waters, USA). Filtered samples were then analyzed using high-performance liquid chromatography (HPLC) by a refractive index detector (RefractoMAX520; ERC, Japan).

Statistical Analysis
All data were expressed as the mean ± SEM. GraphPad Prism (v.8.0) was used to visualize and analyze the data using Tukey's multiple comparison tests. In vitro data were performed using Student's t-test. Statistical significance is denoted as ***p < 0.001, ***p < 0.0001.

L. lactis LB 1022 Suppresses the Production of NO Induced by LPS in RAW264.7 Cells
To evaluate the cell viability effect of L. lactis LB 1022, RAW 264.7 cells were incubated with L. lactis LB 1022 at the determined concentrations for 24 h. L. lactis LB 1022 was not cytotoxic to RAW 264.7 cells tested at 1 × 10 8 CFU with the MTT assay ( Fig. 1). Next, to investigate the inflammatory effects of L. lactis LB 1022, the NO production by RAW264.7 cells due to LPS was assessed as an in vitro model. The results indicated that LPS significantly increased the NO production in RAW 264.7 cells (****p < 0.0001 vs. LPS), whereas L. lactis LB 1022 treatment significantly decreased (****p < 0.0001 vs. LPS; Fig. 1B).

L. lactis LB 1022 Exhibits Anti-Allergy and Anti-Inflammatory Activity
To examine whether L. lactis LB 1022 has anti-allergic activity, the production of β-hexosaminidase was monitored. Untreated and pre-treated with L. lactis LB 1022 for 30 min were challenged with activator C48/80 (Sigma-Aldrich). The stimulated HMC-1 cells treated with L. lactis LB 1022 had β-hexosaminidase levels that were 73.7 ± 0.5% higher than that of the control ( Fig. 2A). To determine whether L. lactis LB 1022 had a regulatory effect on the degranulation of mast cells, the activity of histamine was tested. The results revealed that L. lactis LB 1022 significantly reduced the release of histamine (Fig. 2B). In addition, L. lactis LB 1022 suppressed cytokine levels of IL-4, IL-5, IL-13, and TARC in HaCaT cells (Fig. 2C), indicating that L. lactis LB 1022 inhibited TARC activation, leading to the downregulation of Th2 cytokines.

L. lactis LB 1022 Treatment Alleviates Clinical AD Symptoms in OVA-Induced AD Mice
The animal experimental process is illustrated in Fig. 3A. L. lactis LB 1022 mice group significantly reduced skin symptoms in OVA-induced AD mice model (Fig. 3B). The dermatitis scores in the L. lactis LB 1022 group were significantly decreased than OVA group (***p = 0.0008 at 12 weeks; ****p < 0.0001 at 16 weeks) (Fig. 3C). Itching behaviour was also monitored, and it was found that repeated topical application of L. lactis LB 1022 decreased the itchiness scores compared with OVA group after 12 weeks (***P = 0.0008), with a 2-fold decrease after 16 weeks (***p = 0.0002; Fig. 3D). Furthermore, total IgE was promoted in the OVA group compared with the control and L. lactis LB 1022 groups (Fig. 3E), which suggests that L. lactis LB 1022 leads to the inhibition of AD-like skin symptoms and AD pathology in OVA-induced AD mice.

L. lactis LB 1022 Treatment Alleviates Atopic Dermatitis Symptoms by Decreasing Mast Cell and Eosinophils Infiltration and Regulatory Epithelium-Derived Innate Cytokines in the Skin
OVA-induced mice exhibited AD-like lesions and hyperkeratosis in the skin. Oral administration of L. lactis LB 1022 decreases epidermal thickness (Fig. 4A). To evaluate the skin lesions in more detail, mast cells and eosinophils were stained with Toluidine Blue and H&E, respectively. The count of mast cells and eosinophils was significantly lower in the L. lactis LB 1022 group compared with OVA group (Figs. 4A-4D). Furthermore, the levels of TSLP and TARC were significantly lower in the L. lactis LB 1022 group (Figs. 4E and 4F). Interestingly, the mRNA levels of IL-10 and TGF-β were upregulated by L. lactis LB 1022 treatment (Figs. 4F and 4H). Thus, L. lactis LB 1022 suggested that TSLP and TARC are key players in the AD-like skin phenotype following treatment.

L. lactis LB 1022 Treatment Upregulated the SCFA Concentrations in OVA-Induced Mice
The probiotic strain, L. lactis, is used as a fermentation substrate by gut microbiota-derived metabolites, including acetic acid, butyric acid, and propionic acid. As shown in Fig. 6, L. lactis LB 1022 treatment remarkably   The concentration of (A) acetate, (B) butyrate, (C) propionate, (D) valerate, and (E) lactate measured using HPLC in AD mice. Statistical differences between the OVA and Control/L. lactis LB 1022 groups were assessed using one-way ANOVA followed by Turkey's post-hoc tests (mean ± SEM).

Discussion
The bacterium L. lactis is widely used in dairy products and is extensively used to be a safety probiotic strain, with the discovery of new strains with starter culture properties further expanding its range of industrial applications [7]. In addition, various strains of L. lactis have recently been reported to show anti-inflammatory activity in vitro [16]. For example, oral administration of L. lactis ML2018 remarkably suppressed the expression of inflammatory factors in DSS-induced IBD mice [17]. However, while L. lactis has been widely studied in general, several studies have reported the potential effects of its strains on allergic disease. In this study, we isolated a new probiotic LB 1022 strain from natural cheese with probiotic properties, such as acid and bile tolerance and adherence. LB 1022 exhibited stronger anti-allergy and inflammatory characteristics than the control and was orally administered to an OVA-induced AD mice model to investigate whether it can alleviate AD-like lesions.
Previous studies have shown that dysbiosis of the intestinal microbiota leads to the development of atopic diseases by affecting Th2-related immune responses [18,19]. Administration of L. lactis LB 1022 improved clinical AD symptoms, decreased serum IgE and suppressed the Th2 cytokines secretion, such as IL4, IL-13, and TSLP in blood, which are factors known to be elevated by AD. In our previous study, the oral administration of Lactococcus strains suppressed clinical phenotypes, improved gut microbial-derived metabolites, and regulated Th1/Th2 cytokines in AD mice [20,21]. Similar to these results, L. lactis LB 1022 may have a protective effect against AD by reducing high IgE serum levels and Th2-related responses that arise from an imbalance in the gut microbiota.
AD is an inflammatory disease with complex pathogenesis that includes immune dysregulation. Innate immunity mechanisms, such as the regulation of Th1, Th2, Th17, and Treg, play a central role in the development of AD. In the present study, L. lactis LB 1022 suppressed cytokine levels associated with the Th2 (IL-4, IL-5, and IL-13), Th1 (IL-12 and IFN-γ), and Treg (IL-10) immune response, while also suppressing the production of TSLP/ CCL17 and eotaxin/CCL11 compared with OVA group. Moreover, mRNA expression levels of TSLP and TARC were lower in the dorsal skin. Skin lesions in AD pathogenesis exhibit biphasic inflammation with initial Th2 and chronic Th1 phases [22]. In the initial stage of tissue inflammation, the Th2 immune response contributes to the secretion of IL-4, IL-5, and IL-13 with increased IgE levels [23]. IL-4 plays a crucial role in the pathogenesis of AD via Th2 immunity, IgE switch, and mast cell and eosinophil recruitment, which are involved in itching, dryness and erythema during allergic antigens exposure [24].
Th1 immune responses, including IFN-γ and IL-12, are higher in chronic AD symptoms, with elevated Th2 cytokine levels in acute AD lesions [23]. In addition, Tregs are closely associated with immune suppression and are essential to the control of allergic responses [25]. In the allergic response, Tregs migrate from inflammatory tissue to draining lymph nodes and regulate the inhibition of the Th2 immune response [26]. Consequently, our results suggest that L. lactis LB 1022 ameliorates the symptoms exhibited by OVA-induced AD mice by alleviating the imbalance in Th1/Th2 and suppressing allergic inflammation.
SCFAs, including acetate, butyrate, propionate, valerate, and lactate, are the main metabolites produced in the intestine from the microbial fermented dietary fiber and have a central role in maintaining gut homeostasis and the gut barrier [27]. Compromised epithelial barrier integrity has been reported to be associated with diseases such as AD, asthma, and autoimmunity. Furthermore, SCFAs have been identified to have anti-inflammatory abilities, including promoting Tregs expression [28]. Previously, we reported that OVA-induced gut dysbiosis is associated with AD by reducing SCFA production. [29]. Similarly, our results showed that OVA-induced AD mice had lower SCFA production, which recovered with the administration of L. lactis LB 1022. These results indicate that L. lactis LB 1022 can improve SCFA production and is related to the regulation of allergic responses induced by Treg and Th2 immunity. This study highlights the potential feature of L. lactis as a functional ingredient to ameliorate allergic dermatitis symptoms by promoting gut microbiota-derived metabolites.

Authors Contributions
WK conceived and designed the study. WK and J-HK carried out the statistical analyses. JB and J-HK performed the experiments and drafted the manuscript, with major input from WK.